Genetic information is stored not only in the sequential arrangement of four nucleotide bases, but also in covalent modification of selected bases (see, e.g., Robertson et al., Nature Rev. Genet. 1:11-19 (2000)). One of these covalent modifications is methylation of cytosine nucleotides, particularly cytosines adjacent to guanine nucleotides in “CpG” dinucleotides. Covalent addition of methyl groups to cytosine within CpG dinucleotides is catalyzed by proteins from the DNA methyltransferase (DNMT) family (Amir et al., Nature Genet. 23:185-88 (1999); Okano et al., Cell 99:247-57 (1999)). In the human genome, CpG dinucleotides are generally under represented, and many of the CpG dinucleotides occur in distinct areas called CpG islands. A large proportion of these CpG islands can be found in promoter regions of genes. The conversion of cytosine to 5′-methylcytosine in promoter associated CpG islands has been linked to changes in chromatin structure and often results in transcriptional silencing of the associated gene. Transcriptional silencing by DNA methylation has been linked to mammalian development, imprinting and X-Chromosome inactivation, suppression of parasitic DNA and numerous cancer types (see, e.g., Li et al., Cell 69:915-26 (1992); Okano et al., Cell 99:247-57 (1999)). Detected changes in the methylation status of DNA can serve as markers in the early detection of neoplastic events (Costello et al., Nature Genet. 24:132-38 (2000)).
Studies demonstrating the practical use of DNA methylation analysis in a clinical environment are scarce. This is due, at least in part, to the technical limitations facing DNA methylation research. A few DNA methylation analysis techniques have been used, but each method has its limitations. See, for example, U.S. Pat. No. 6,214,556 directed to methods for producing complex DNA methylation fingerprints. The methods of this patent amplify fragments of genomic DNA that have been treated with bisulfite using with degenerated oligonucleotides or oligonucleotide that are complimentary to adaptor oligonucleotides that have been ligated to the fragmented genomic DNA. Methods such as these are prone to false positive results and are limited in accurate methylation assessment to a single cytosine position per analysis. Often times they require large amounts of high quality genomic DNA and are labor intensive.
Since DNA methylation has a variety diagnostic uses, there is a need for reliable, cost effective, high throughput DNA methylation analysis tools and methods to evaluate potential methylated sites, to associate methylation sites with disease, and to develop prognostic methylation markers. Therefore it is an object herein to provide methylated nucleotide identification methods, and products therefor.